JPH0795850A - Formulated milk for infant - Google Patents

Abstract

PURPOSE:To obtain a formulated milk for infant, having enhanced effect on bone formation, capable of assisting bone growth of baby growing under artificial nutrient by IGF-1 action, preventing bone diseases such as osteoporosis. CONSTITUTION:This formulated milk for infant is mixed with insulin growth factor-1 (IGF-1). The added amount of IGF-1 is properly 24-1,150mug/100g in the case of powdered milk and 3-150mug/100g in the case of liquid formulated milk.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to infant formula containing insulin-like growth factor-1 to enhance the osteogenic effect. The infant formula of the present invention can grow normal cells such as human osteoblasts by the action of insulin-like growth factor-1 to promote and strengthen robust bone formation in infants.

[0002]

2. Description of the Related Art In recent years, the bones of infants and children have become weak and prone to fracture. In addition, the number of patients with various bone diseases such as osteoporosis, bone fracture and low back pain is increasing with the aging of the population. Insufficient intake of calcium, decreased calcium absorption, activated vitamin D3
Insufficiency of secretion and imbalance of hormones have been pointed out. Therefore, various calcium agents for the purpose of calcium supplementation for preventing these diseases have been developed. Furthermore, in order to prevent bone diseases such as osteoporosis, it has been clarified that it is effective to increase the bone mass during the growth period including infancy so as to increase the maximum bone amount as much as possible. Also in nutrition, various calcium agents such as calcium carbonate have been added to infant formulas for the purpose of strengthening bones. However, it has been reported that calcium contained in milk is poorly absorbed as compared with calcium contained in breast milk (Gu
rr, M. I. J. Dairy Res. , 48th
Vol. 519-554, 1981), in fact, some of the calcium agents added to these infant formulas form insoluble salts in the digestive tract and are not sufficiently absorbed in the body. .

On the other hand, insulin-like growth factor (insulin-lik)
e growth factor) -1 (hereinafter abbreviated as IGF-1)
Has a molecular weight of about 7,800 that belongs to the somatomedin class.
Bovine IGF-1 and human IGF-1 are known to have the same structure (GL Francis et al., Biochem. J.,
251, pp. 95-103, 1988). Many methods for producing human IGF-1 by gene recombination are also known, and it is also known that the structure of IGF-1 is exactly the same as that of human IGF-1. Furthermore, IGF-1 is an important factor in bone metabolism and is known to have an action of activating osteoblasts and strengthening bone (Shiro Saito et al., Nihon Rinji, Vol. 48, No. 12, 2779-2784, 1990
Year). Then, it was revealed that a receptor for IGF-1 exists in the digestive tract, and IG orally ingested
It has been suggested that F-1 acts via a gastrointestinal receptor (Kazutoshi Takano, Hormone and Clinic, Vol. 39, No. 12, 31-37, 1991).

[0004]

DISCLOSURE OF INVENTION Problems to be Solved by the Invention The present inventors have made a comparative study on the bone formation of breast-fed infants and artificially fed infants, and have come to recognize that the bone formation of artificially fed infants is not always sufficient. Then, in order to elucidate the factor, when the amount of IGF-1 contained in the commercially available infant formula was analyzed, IGF-1 contained in the breast milk was analyzed.
It was found to be about 40% of the amount. In addition, when a cell proliferation test of breast milk and infant formula was carried out using a cultured osteoblast-like strain (MC3T3-E1), the cell proliferation activity of infant formula was about 70% of that of breast milk. I found that. Then, they found that supplementing IGF-1 lacking in infant formula can promote bone formation in artificially fed infants, and completed the present invention. Therefore, the present invention proliferates normal cells such as human osteoblasts and promotes strong bone formation in infants.
An object of the present invention is to provide infant formula containing -1 for enhancing the bone formation effect.

[0005]

The feature of the present invention is that IGF
-1 was added to infant formula to enhance the osteogenic effect. The infant formulas referred to here are roughly classified into infant formulas and infant formulas, and infant formulas include infant formulas, premature infant formulas, follow-up formulas, and pharmaceutical special formulas. Powdered milk and other milk powders used for artificial feeding of infants are meant, and the liquid infant formula means liquid infant formula prepared for the purpose of artificial infant feeding. Examples of IGF-1 used in the present invention include IGF-1 isolated from breast milk and milk, or IGF-1 produced by gene recombination technology. Practically, IGF-1 isolated from milk
It is preferable to use a high content composition. In addition, when the present inventors measured with a commercially available radioimmunoassay (RIA) kit (manufactured by Amersham), IGF contained in milk was measured.
The content of -1 was 20 ng / ml. As the IGF-1 in the present invention, not only a single compound thereof, but also a high concentration IGF-1 content product obtained from raw milk or whey protein concentrate is used.

The method for separating a composition containing a large amount of IGF-1 from milk will be exemplified below. The supernatant obtained by heating the whey at 85 ° C. for 20 minutes is subjected to ultrafiltration with a molecular fraction of 8,000 to obtain a composition having a high IGF-1 content as a concentrate. About 20 μg / g of IG per dry weight
F-1 is included. Also, skim milk is heated at 121 ° C. for 2 seconds, passed through a cation exchange resin to adsorb IGF-1 onto the resin, and then the resin is thoroughly washed with water to give 1 mol Na.
The IGF-1 fraction adsorbed on the resin is eluted with an aqueous Cl solution. Then, this eluate is subjected to ultrafiltration with a molecular fraction of 8,000 to obtain a composition having a high IGF-1 content as a concentrate. About 200 per dry weight in this concentrate
It contains μg / g of IGF-1. The concentrated or dried IGF-1 high-content composition obtained by these methods can be used as it is for infant formula and infant formula, but if necessary, such as ion exchange chromatography. IGF attached to the fractionation means
It can also be used as a composition containing a high concentration of -1 and having a high IGF-1 content. Further, since IGF-1 is stable against heat treatment, it is possible to heat-sterilize a raw material containing IGF-1 or a modified milk by a commonly used method.

Next, the method of blending IGF-1 will be described. In the case of producing liquid infant formula, a sterile IGF-1 concentrated solution is added to a solution prepared by sterilizing raw materials, mixed, and cooled according to a conventional method to obtain a product. Also,
In the case of infant formula, IGF-1 is added to a solution prepared by homogenizing raw materials, mixed, and concentrated according to a conventional method.
The product can be dried to obtain a product, or the sterilized concentrated liquid containing IGF-1 can be powdered by a means such as freeze-drying, and then powdered with infant formula to give a product.

According to the present invention, the IG compounded in the infant formula
The amount of F-1 was calculated as follows. Usually, the intake of breast milk or infant formula for immature infants is about 70 ml / day per 1 kg of body weight. When converted into IGF-1 intake per kg of body weight, about 7 μg / day for breast-fed infants and about 3 μg / day for artificially fed infants.
As a result, artificial feeding infants will have an insufficient intake of IGF-1 of about 4 μg / day. In addition, since it is said that normal infants ingest up to 250 ml / day of breast milk or infant formula per 1 kg of body weight, artificial feeding infants lack about 15 μg / day of IGF-1 intake compared with breast-fed infants. Becomes
Therefore, IGF-1 may be added to infant formula in order to supplement these shortages of IGF-1 intake. Cultured osteoblast-like strain (MC3T3-E1) performed by the present inventors
According to the result of the cell proliferation test using the above, in order to obtain the cell proliferation effect equivalent to that of the milk, the average IGF-1 concentration of the milk was 7
It was also clarified that 0% or more is required, and even if 1700% or more is added, no further cell growth effect can be obtained. In consideration of these, IGF-1 to be blended in the present invention to impart the same effect as that of breast milk to infant formula.
The amount of solid is 13 to 16% by weight when preparing infant formula, and in the case of infant formula, it is 24 to 1,150 μ.
g / 100g, 3 to 150μ for liquid infant formula
Based on g / 100 ml.

Milk, milk and infant formula solution 100μ
Acid-ethanol solution (solution in which 87.5% ethanol and 12.5% 2N hydrochloric acid are mixed) 40
It was dissolved in 0 μl, stirred well, and then incubated at room temperature for 30 minutes. Then, it was centrifuged at 1,860 × g to obtain 100 μl of the supernatant. 100 μl of this supernatant was added to 0.
After adding 40 μl of 855 mol of Tris base and thoroughly stirring, 1,260 μl of a measurement buffer (RIA kit manufactured by Amersham Co.) was further added and well stirred to obtain a total amount of 1,4
00 μl of measurement sample was obtained. This sample 100
Using μl, measurement was carried out with a RIA kit (manufactured by Amersham).

Next, the present invention will be specifically described with reference to Reference Examples and Examples.

[Reference Example 1] 10% of commercially available whey protein concentrate (WPC)
Solution that is sufficiently dissolved in distilled water so that the concentration becomes
6.8) after heating 40 l at 85 ° C. for 20 minutes,
Centrifugation at 000 × G gave a supernatant. The supernatant is then ultrafiltered with a molecular fraction of 8,000 and concentrated,
It was freeze-dried to obtain 131 g of a powder having a high content of IGF-1. When the concentration of IGF-1 contained in this powder was measured by RIA, it was 23 μg / g.

[0011]

[Reference Example 2] 40 l of raw bovine milk was sterilized by heating at 121 ° C for 2 seconds, and then this sterilized milk was packed with a cation exchange resin (trade name: sulfonated chitopearl, manufactured by Fuji Spinning Co., Ltd.) (diameter). 5 cm x 25 cm in height) 15 ml /
The solution was passed through at a flow rate of minutes to adsorb IGF-1 onto the resin. Next, this column was thoroughly washed with deionized water, and then the IGF-1 fraction was eluted with 1 molar NaCl, and this eluted fraction was subjected to ultrafiltration and diafiltration with a molecular fraction of 8,000. The mixture was desalted, concentrated, and lyophilized to obtain 85 g of IGF-1 high-containing powder. IGF-1 contained in this powder
When the concentration was measured by RIA, it was 213 μg / g.

[0012]

[Reference Example 3] IGF-obtained by the method of Reference Examples 1 and 2
1 IGF-1 content was measured by RIA for high content powder, breast milk, milk, and commercially available infant formula. The results are shown in Table 1. The IGF-1 high content powder and the commercially available infant formula were dissolved in pure water so that the solid content was 13%.

[0013]

[Table 1] ──────────────────────────────── Sample IGF-1 concentration (μg / ml) ─── ───────────────────────────── Breast milk 0.11 Raw bovine milk 0.02 Commercial infant formula 0.04 Reference example 1 IGF-1 high content powder of 2.98 IGF-1 high content powder of Reference Example 2 27.64 ──────────────────────────── ─────

[0014]

Example 1 Whey protein concentrate 15.9 kg, lactose 45.
9kg, vitamins and minerals 1kg, water 650k
After being dissolved in g, it was mixed with 257 kg of desalted skim milk, 7 kg of fresh cream and 23.9 kg of vegetable oil were added, mixed and homogenized, and the resulting solution was sterilized by a conventional method to prepare a raw material solution. did. On the other hand, 2.5 kg of IGF-1 high content powder prepared by the method shown in Reference Example 1 was dissolved in 200 kg of sterilized water, and then sterilized with a sterilization filter I
A GF-1 solution was prepared. Then, these solutions were mixed and then spray-dried to obtain 120 kg of infant formula. The infant formula thus obtained has 7
It contained 7.3 μg / 100 g of IGF-1.

[0015]

Example 2 Whey protein concentrate 15.9 kg, lactose 45.
9kg, 1kg vitamins and minerals 352k water
After dissolving in g, it was mixed with 257 kg of desalted skim milk, 7 kg of fresh cream and 23.9 kg of vegetable oil were added, mixed and homogenized, and the obtained solution was sterilized according to a conventional method to prepare a raw material solution. On the other hand, 0.2 kg of high IGF-1 content powder prepared by the method shown in Reference Example 2 was added to 20 kg of sterilized water.
Then, IGF-1 solution that had been sterilized with a sterilization filter was prepared. Then, these solutions were mixed to obtain 720 kg of infant formula. The liquid infant formula thus obtained contains 10.5 μg / 100 ml of I
GF-1 was included.

[0016]

[Test Example 1] Breast milk, commercially available infant formula, IGF-1 high-content powder obtained by the method of Reference Examples 1 and 2, commercially available infant formula obtained by the method of Reference Example 1 or 2. I
Regarding infant formula powder containing GF-1 high content powder,
The effect of promoting the proliferation of osteoblasts was examined. The cultured osteoblast-like strain (MC3T3-E1) was removed into a 96-well flat-bottom cell culture plate, and cultured in an α-MEM medium containing 0.3% bovine serum. To 100 μl of the medium, add 2 μl of each of the following samples dissolved in 0.5% concentration, and
It was cultured for 8 hours. In addition, except for breast milk, it was dissolved in pure water so as to have a solid content of 13% and subjected to the test. After that, thymidine labeled with tritium was added, and after 2 hours, the radioactivity of thymidine taken up by the cells was measured to determine the proliferative activity of osteoblasts. This activity is 0.3
The radioactivity of thymidine incorporated into cells when cultured only in α-MEM medium containing 100% bovine serum was defined as 100%, and the radioactivity when each sample was added and cultured was calculated and shown. The results are shown in Table 2.

Sample 1: Breast Milk Sample 2: Commercial Infant Formula Milk Powder Sample 3: IGF-1 High Content Powder (Reference Example 1) Sample 4: Commercial Infant Formula Powder Milk 1.0 g + IGF-1 High Content Powder (Reference Example 1) 0.008 g Sample 5: Commercially available infant modified milk powder 1.0 g + IGF-1 high content powder (Reference Example 1) 0.01 g Sample 6: Commercially available infant modified milk powder 1.0 g + IGF-1 high content powder (Reference Example 1) 0.013 g Sample 7: Commercially available baby Formulated milk powder 1.0 g + high IGF-1 content powder (Reference Example 1) 0.02 g Sample 8: Commercial infant formula powder 1.0 g + IGF-1 high content powder (Reference Example 1) 0.04 g Sample 9: Commercial infant formula powder 1.0 g + IGF- 1 High content powder (reference example 1) 0.06 g Sample 10: Commercial infant formula powder 1.0 g + IGF-1 high content powder (reference example 1) 0.07 g Sample 11: Commercial infant formula powder 1.0 g + IGF-1 high content powder (reference Example 1) 0.09 g Sample 12: Commercial infant formula 1.0 g + IG -1 High content powder (Reference example 1) 0.20 g Sample 13: Commercial infant formula powder 1.0 g + IGF-1 high content powder (Reference example 1) 1.0 g Sample 14: Commercial infant formula powder 1.0 g + IGF-1 high content powder ( Reference Example 1) 1.2 g Sample 15: IGF-1 high content powder (Reference Example 2) Sample 16: Commercial infant formula powder 1.0 g + IGF-1 high content powder (Reference Example 2) 0.0009 g Sample 17: Commercial infant formula powder 1.0 g + IGF-1 high content powder (reference example 2) 0.001 g Sample 18: Commercial infant formula powder 1.0 g + IGF-1 high content powder (reference example 2) 0.0015 g Sample 19: Commercial infant formula powder 1.0 g + IGF-1 high content Powder (Reference Example 2) 0.002 g Sample 20: Commercial infant formula 1.0 g + IGF-1 high content powder (Reference Example 2) 0.004 g Sample 21: Commercial infant formula 1.0 g + IGF-1 high content powder (Reference Example 2) 0.006 g Sample 22: Commercially available infant formula 1.0 g + IGF-1 high content powder (Reference Example 2) 0.0 07 g Sample 23: Commercial infant formula powder 1.0 g + IGF-1 high content powder (Reference Example 2) 0.008 g Sample 24: Commercial infant formula powder 1.0 g + IGF-1 high content powder (Reference Example 2) 0.015 g Sample 25: Commercial Infant formula 1.0 g + IGF-1 high content powder (reference example 2) 0.058 g Sample 26: Commercial infant formula 1.0 g + IGF-1 high content powder (reference example 2) 0.062 g

[0018]

[Table 2]

From the above test results, the infant formula prepared by adding IGF-1 to the commercially available infant formula at a concentration of 70% or more of the mother formula has the same cell proliferation ability as that of the mother formula. It was found that they have an osteoblast proliferation promoting action.
However, when the amount of IGF-1 blended is less than 70% of breast milk, only the same level of cell proliferation ability as that of commercially available infant formula is obtained, and when IGF-1 is added in excess of 17 times the amount of breast milk. However, it was found that there was no change in cell proliferation ability.

[0020]

EFFECT OF THE INVENTION IGF incorporated into the infant formula of the present invention
Since -1 has an effect on bone strengthening, it can assist the bone growth of artificially fed infants, and further prevent various bone diseases thought to occur in association with this, particularly osteoporosis. Further, by using the milk-derived IGF-1, it becomes possible to provide at low cost infant formula which has no problem in terms of safety.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Hiroaki Matsuyama 5-11-3 Shinjuku-cho, Kawagoe-shi, Saitama (72) Inventor Hiroshi Kawakami 2-6-13-501 (72) invention Tsurugaoka, Oi-cho, Iruma-gun, Saitama Person Masahiro Yahiro 2-8-13 Kumegawa-cho, Higashimurayama-shi, Tokyo

Claims (5)

[Claims] 1. An infant formula containing insulin-like growth factor-1 to enhance the osteogenic effect. 2. The formula according to claim 1, wherein the infant formula is infant formula. 3. Insulin-like growth factor-1 24-1,
The modified milk according to claim 2, which is blended at a ratio of 150 μg / 100 g. 4. The infant formula according to claim 1, wherein the infant formula is a liquid infant formula. 5. Insulin-like growth factor-1 from 3 to 150
The modified milk according to claim 4, which is blended at a ratio of μg / 100 ml.